Lipoproteins are complex particles consisting of protein and lipid which are found in the circulatory system. One of the functions of lipoproteins is to carry water-insoluble substances such as cholesterol and cholesterol esters for eventual cellular use. While all cells require cholesterol for growth, excess accumulation of cholesterol by cells is known to lead to certain diseases, including atherosclerosis.
It is known that the amount of total serum cholesterol can be correlated with the incidence of atherosclerosis. However, there are a variety of classes of lipoproteins in serum which can be classified by their density. These classes include very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). All of these classes of lipoproteins contain varying amounts of cholesterol, and a total serum cholesterol determination is a complex average of the amount that each lipoprotein-class contributes to the total lipoprotein population of the serum.
It has long been suspected that some lipoprotein classes are more closely associated than other lipoprotein classes with the progression of heart disease, including atherosclerosis. In fact more recent studies have implicated LDL as the class of lipoproteins responsible for the accumulation of cholesterol in cells, whereas HDL has been shown to be important in the removal of excess cholesterol from cells. Additionally the correlation of atherosclerosis and the levels of LDL cholesterol is much higher than a similar correlation between atherosclerosis and total serum cholesterol levels. Conversely, there appears to be a negative correlation between atherosclerosis and HDL cholesterol levels.
Despite the desirability of differentiating LDL cholesterol levels in blood plasma from those of other soluble cholesterols, a technique suitable for use in clinical laboratories has not heretofore existed. One method which has been suggested relies upon the interaction of heparin in the presence of calcium to precipitate both LDL and VLDL, cf. Bursterin et al., Adv. Lipid. Res. 11:67 (1973). To separate the LDL and VLDL fractions, ultracentrifugation techniques, which are time consuming and expensive, may be used. Ultracentrifugation, to separate the lipoproteins solely on the basis of their density, requires special equipment and long processing time. Electrophoretic separation also requires special equipment and long processing times.
A variety of precipitation methods have been used which depend upon the use of polyanions and divalent cations, Okabe, Xth Int. Cong. of Clin. Chem., Mexico (1978); Genzyme Diagnostic, Cambridge, Mass., LDL cholesterol precipitation reagent package insert. Other precipitation methods use polymers, as shown in U.S. Pat. No. 4,474,898 and U.S. Pat. No. 4,647,280; or lectin, as disclosed in U.S. Pat. No. 4,126,416. Kerscher et al., in U.S. Pat. No. 4,746,605, teach that VLDL and HDL can be precipitated by HDL antibodies with polyanions and divalent cations. However, the amount of antibodies required with this method is too expensive for routine use.
Other methods for determining the amount of lipoprotein fractions in samples are known, but these methods are not suitable for use in a dry-chemistry device which can be used for simple and rapid determination of lipoproteins. For example, Pascal, in U.S. Pat. No. 4,366,244, discloses that lipoprotein fractions can be separated by using a lectin to separate the LDL and VLDL fractions, and then measuring the amount of cholesterol in the precipitate and in the remaining solution. This method requires centrifugation of the precipitate, and measurement of both the precipitate and the remaining solution. Ziegenhorn et al., in U.S. Pat. No. 4,486,531, disclose a turbidimetric process for detection of beta-lipoproteins (LDL) in body fluids by precipitation with polyanions and divalent cations. The LDL then can be detected directly by a turbidimetric determination.
When LDL is precipitated with polyanions such as dextran sulfate and divalent cations such as magnesium, the precipitate redissolves if one tries to selectively convert the cholesterol in the supernatant by an enzymatic assay which requires the presence of surfactants. Moreover, cholesterol esterase and cholesterol oxidase present in the system hydrolyze the LDL.
Another method for determining LDL is calculation by the Friedewald Formula, as disclosed in Friedewald et al., Clin. Chem. 18: 499-502 (1972). In this method, LDL is estimated by the total cholesterol, HDL, and triglyceride contents of the sample. This method requires multiple assays, and is not accurate for samples containing high levels of triglycerides.
Consequently, there is a need for a simple procedure or device for the determination of LDL lipoprotein accurately.